准分子激光角膜切削术（PRK）后大鼠角膜中转化生长因子（TGF）及细胞外基质蛋白的mRNA测量

Measurement of mRNAs for TGFss and extracellular matrix proteins in corneas of rats after PRK.

作者信息

Chen C, Michelini-Norris B, Stevens S, Rowsey J, Ren X, Goldstein M, Schultz G

机构信息

Institute of Wound Research, Department of Obstetrics and Gynecology University of Florida, Gainesville, USA.

出版信息

Invest Ophthalmol Vis Sci. 2000 Dec;41(13):4108-16.

PMID:11095603

Abstract

PURPOSE

To assess the role of the transforming growth factor (TGF)ss system in formation of corneal haze after excimer laser photorefractive keratectomy (PRK), levels of mRNAs for three TGFss isoforms (TGFss1, TGFss2, and TGFss3), the TGFss type II receptor (TssRII), and extracellular matrix (ECM) genes including fibronectin (FN), collagen I, collagen III, and collagen IV were measured in rat corneas.

METHODS

Corneas were graded for corneal haze at 0, 1.5, 7, 21, 42, and 91 days after PRK. Total RNA was isolated from pooled corneas, and the levels of mRNAs were measured using competition-based quantitative reverse transcription-polymerase chain reaction (RT-PCR).

RESULTS

Severe corneal haze developed by day 42 and persisted to day 91. Levels of TGFss1 mRNA were high in rat corneas before PRK and remained relatively constant. In contrast, levels of TGFss2 and TGFss3 mRNAs were very low in normal corneas, increased 300-fold and 25-fold, respectively, on day 21, and remained elevated on day 91. Levels of mRNA for TssRII increased, with a peak elevation of 50-fold on day 42 after PRK. Levels of mRNAs for ECM proteins also increased. Fibronectin mRNA was nondetectable in normal corneas but rapidly increased to 675 copies/cell on day 7 and remained elevated to day 91. Collagen III mRNA levels peaked on day 21 with a 700-fold increase compared with a very low level of expression in normal cornea, and then decreased on day 91. Expression of collagen I mRNA lagged expression of collagen III mRNA and peaked at day 42 after PRK with a 1200-fold increase over normal cornea. In contrast, mRNA for collagen alpha(1)IV, a major component in basement membranes, remained relatively stable through day 21 and then increased slightly on days 42 and 91.

CONCLUSIONS

The synchronized increase in mRNA synthesis for both the TGFss system and key ECM genes supports the hypothesis that TGFss is a key growth factor promoting stromal haze formation in corneas after PRK and suggests that limiting TGFss system may reduce corneal scarring after excimer laser ablation.

摘要

目的

为评估转化生长因子（TGF）β系统在准分子激光屈光性角膜切削术（PRK）后角膜混浊形成中的作用，测定大鼠角膜中三种TGFβ亚型（TGFβ1、TGFβ2和TGFβ3）、TGFβⅡ型受体（TβRⅡ）以及包括纤连蛋白（FN）、Ⅰ型胶原、Ⅲ型胶原和Ⅳ型胶原在内的细胞外基质（ECM）基因的mRNA水平。

方法

在PRK术后0、1.5、7、21、42和91天对角膜的混浊程度进行分级。从收集的角膜中提取总RNA，使用基于竞争的定量逆转录-聚合酶链反应（RT-PCR）测定mRNA水平。

结果

到第42天时出现严重的角膜混浊，并持续至第91天。PRK术前大鼠角膜中TGFβ1 mRNA水平较高且保持相对稳定。相比之下，正常角膜中TGFβ2和TGFβ3 mRNA水平非常低，在第21天时分别增加了300倍和25倍，并在第91天时仍保持升高。TβRⅡ的mRNA水平升高，在PRK术后第42天达到峰值升高50倍。ECM蛋白的mRNA水平也升高。正常角膜中未检测到纤连蛋白mRNA，但在第7天时迅速增加至675拷贝/细胞，并在第91天时仍保持升高。Ⅲ型胶原mRNA水平在第21天达到峰值，与正常角膜中极低的表达水平相比增加了700倍，然后在第91天时下降。Ⅰ型胶原mRNA的表达滞后于Ⅲ型胶原mRNA的表达，在PRK术后第42天达到峰值，比正常角膜增加了1200倍。相比之下，基底膜的主要成分Ⅳ型胶原α(1)mRNA在第21天前保持相对稳定，然后在第42天和第91天时略有增加。